1. Field of Invention
This invention relates to an insulin-like growth factor binding protein acid to the means and methods for its production in therapeutically significant quantities.
2. Description of the Background Art
Peptides of the insulin-like growth factor (IGF) family, the recombinant production of which is described, e.g., in EP 128,733 published Dec. 19, 1984, to Lee et al., are found associated with binding proteins in the circulation, in other body fluids, and in media conditioned by cultured cells. Two IGF binding proteins, found in different fluids, have been identified and characterized. Hintz, R. L. (1984) Clinics in Endo. and Metabol., 13: 31-42; Martin, J. L. and Baxter, R. C. (1986) J. Biol. Chem., 261: 8754-8760; Povoa et al. (1984) Eur. J. Biochem., 144: 199-204. One predominates in adult serum, while the other is found in highest concentrations in amniotic fluid. Baxter et al. (1987) J. Clin. Endo. and Metabol., 65: 423-431.
The levels of the binding protein in adult serum have been found to reflect the growth hormone (GH) status of individuals who are either GH-deficient or acromegalic. Thus, high levels of binding protein correlate with high levels of GH. Martin and Baxter (1985) J. Clin. Endo. and Metabol., 61: 799-801. Thus, this binding protein has been referred to as the GH-dependent IGF binding protein. For convenience, the designations BP53 for the GH-dependent binding protein from human plasma and BP28 for the binding protein first isolated from amniotic fluid are used herein. The designations indicate the size of the purified proteins on non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
BP53 is found as an acid-stable component of a 125-150 kD glycoprotein complex contained in human plasma that carries most of the endogenous IGFs and is also regulated by GH. White et al. (1981) J. Clin. Endo. and Metabol., 53: 49ff; Hintz et al., J. Clin. Endo. and Metabol., 53: 100ff; and Daughaday et al. (1982) J. Clin. Endo. and Metabol., 55: 916ff. The binding component of this complex, purified after acidification, has a molecular weight on SDS-PAGE of 43 kD (reduced) and 53 kD (non-reduced). The purified protein has a high affinity for IGF-I and IGF-II (K.sub.a =20-40 nM.sup.-1). Martin and Baxter, J. Biol. Chem., op cit. The concentration of BP53 in normal adult plasma is 6.1 mg/l. The levels of BP53 are increased in acromegalic subjects to 13.5 mg/l and decreased in GH-deficient subjects to 2-3 mg/l. Baxter and Martin, J. Clin. Invest., 78: 1504-1542 (1986). While BP28 predominates in amniotic fluid, BP53 is also found in amniotic fluid at about 4.6 mg/l. The rat homolog of BP53 has been purified, and it has considerable N-terminal amino acid sequence homology with the human protein. Baxter and Martin (1987) Biochem. Biophys. Res. Comm., 147: 408-415.
BP28 has a molecular weight of 35-40 kD as determined by gel filtration chromatography (Drop et al. (1984) J. Clin. Endo. and Metabol., 59: 899ff), similar to the 32 kD (reduced) or 28 kD (non-reduced) molecular weight found on SDS-PAGE. The purified protein binds both IGF-I and IGF-II, but with a lower affinity than BP53 (Ka=3-7 nM.sup.-1). Concentrations from 37 to 148 mg/l have been reported by Baxter et al., supra, for BP28 in amniotic fluid. While BP53 predominates in adult serum, BP28 is present in serum where its concentrations range from 0.02 to 0.35 mg/l with a marked diurnal cycle peaking at 0600 to 0800 hours. Baxter and Cowell (1987) J. Clin. Endo. and Metabol., 65: 432-440. No such diurnal variation has been found for BP53. N-terminal amino acid sequence data show that two other human proteins, PP12 isolated from placenta (Koistinen et al. (1986) Endocrin., 118: 1375ff) and an IGF binding protein isolated from the culture medium of a hepatoma cell line (HEP-G2) (Povoa et al. (1985 ) Biochem. Biophys. Res. Comm., 128: 1071ff), are the same or closely related to BP28. An IGF binding protein has also been isolated from the culture medium of the rat cell line, BRL-3A. Lyons and Smith (1986) Mol. Cell. Endo., 45: 263-270; Mottola et al. (1986) J. Biol. Chem., 261: 11180-11188. Based on similar size properties, high fetal serum concentration, and some protein sequence similarity, it is likely that this IGF binding protein is the rat homolog of BP28.
Although the isolation and purification of BP53 has been described in the literature as shown above, the relatively low concentration of the protein in human plasma and the high cost, both in terms of effort and expense, of recovering in commercial quantities purified protein from the plasma hinder its broad-scale use, either alone or in combination with IGF.
Accordingly, it is an object of the present invention to isolate DNA encoding BP53 and to produce commercially useful quantities of the protein from a therapeutically acceptable source.
It is a further object to obtain BP53 in a form unaccompanied by the glycosylation associated with the native BP53.
It is an additional object to prepare amino acid sequence and other variants of BP53 that do not substantially adversely affect the biological activity of the protein.
It is yet another object to produce BP53 completely free of other naturally occurring (source) proteins.
These and other objects of the invention will be apparent from the specification as a whole.